This invention relates in general to molecular biology, pharmacology and medicine and in particular to the isolation, purification and cloning of a human proteinase inhibitor.
Preservation of the integrity of local organ function requires a delicate balance of the activities of phagocytic cell proteinases and the action of proteinase inhibitors. Loss of this balance is believed to be a major causative factor in the pathogenesis of asthma, chronic bronchitis, cystic fibrosis, emphysema, sarcoidosis, respiratory distress syndromes, arthritis, and certain skin diseases and possibly malignancies. For example, excess release of elastase by neutrophils and monocytes as well as excess accumulation of monocytes and neutrophils are believed responsible for tissue injury in inflamatory conditions such as arthritis and emphysema and in neutrophil mediated injury to endothelial cells. To acquire the ability to monitor and manipulate the proteinase-proteinase inhibitor balance requires that the relevant molecules be identified, isolated and purified.
Of the phagocytic cell proteinases, an important one is the serine active site proteinase that is commonly called "neutrophil elastase". Human neutrophil elastase is a 218 amino acid glycosylated protein of known sequence that is particularly abundant in neutrophils (0.5% of total protein) and is also found in monocytes and macrophages. Elastase cleaves extracellular matrix proteins such as elastin, proteoglycans, fibronectin, type III and type IV collagen, and certain soluble proteins. It also is required by neutrophils for their migration through cell barriers in vitro.
The continuous action of elastase inhibitors in vivo is evident from the neutrophil turnover rate. Despite the fact that neutrophils enter most body sites, turnover of about 10.sup.11 neutrophils with a content of about 50 mg elastase occurs daily in humans without evidence of uncontrolled tissue degradation.
A prevalent soluble blood protein, .alpha.l-antitrypsin (.alpha.l-AT), is a fast-acting elastase inhibitor in vitro. Individuals with genetically reduced levels of .EPSILON.l-AT (homozygous Z-variant) are predisposed to develop pulmonary emphysema in the third or fourth decade of life due to uncontrolled elastase action. Human .alpha.l-AT currently is used to treat congenital .alpha.l-AT deficiency.
Molecules differing from .alpha.l-AT that fulfill the requirements of a physiological regulator of neutrophil elastase activity have been detected in monocytes and neutrophils in several species. It was reported in 1971 that an endogenous elastase inhibitor with properties of a protein was detected in the cytosolic fraction of human blood leukocytes and human lung macrophages (1,2). Cytosolic proteins that inhibit elastase were identified and purified from horse blood neutrophils (3,4), pig blood leukocytes (5) and bovine lung macrophages (6). An elastase inhibitor in the extracellular fluid of cultured guinea pig macrophages has been identified by its ability to form a covalent complex with elastase (7). Larger quantities of the guinea pig elastase inhibitor have been found in macrophage lysates (7). More recently, a prevalent, fast-acting endogenous elastase inhibitor protein has been detected in mature human monocytes and monocyte-like cells (8). To date, no one has been able to isolate, purify, characterize and clone this human elastase inhibitor.